Research Theses
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This collection contains Ph.D. and Masters (Research) theses from University of Adelaide postgraduate students.
Note that in many cases the full content of the thesis is not available here; instead we have scanned the title page, contents pages and abstract from each thesis and made that available as a PDF file. In 2015, the Library began a project to digitise and make all research theses available online. We expect new theses to be deposited here in complete form. Older theses may be included here on request from the author.
Theses listed here will also be included in the National Library of Australia's Trove service.
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Browsing Research Theses by Advisors "Able, Amanda Jane"
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Item Open Access Application of citral to control postharvest diseases of oranges.(2011) Wuryatmo, Erminawati; Scott, Eileen Sandra; Able, Amanda Jane; Ford, Christopher Michael; School of Agriculture, Food and WineGreen mould, blue mould and sour rot, caused by the fungi Penicillium digitatum, P. italicum and Geotrichum citri-aurantii, are postharvest diseases which cause significant losses to the citrus industry worldwide. Current control of the diseases raises some problems, such as development of fungicide resistance, concerns about residues harmful to humans, and also restrictions on the use of certain fungicides. Those problems have led to a need to develop alternative fungicides, including exploitation of some natural products such as essential oils. Application of the essential oil, citral (3,7-dimethyl-2,6-octadienal) to control the fungi and the diseases was assessed in this study. In vitro, citral incorporated into agar at 2%, 6% and 15% prevented germination of spores of the fungi, and no mycelial growth was observed by microscopic observation after 17 days of incubation. When citral was applied as a solution on agar, spore germination of P. digitatum and G. citri-aurantii was inhibited at concentrations of 6% and 15%. However, germination of P. italicum spores was not affected. Vapour of citral and its individual isomers, geranial and neral, generated from 6 and 15% aqueous solutions, inhibited spore germination and growth of the three pathogens. Vapour generated from 15% aqueous solutions of citral and geranial were fungicidal to P. digitatum and G. citri-aurantii, and fungistatic to P. italicum, while neral was fungicidal to G. citri-aurantii and fungistatic to the other two fungi. The result suggested that method of application and citral concentration affected the efficacy of citral in controlling the fungi. In the three methods of applications examined, citral was effective in controlling G. citri-aurantii, especially at high concentration. As an α , β-unsaturated aldehyde, citral may be degraded over time due to oxidative reactions, resulting in change in its composition, and this may affect its antifungal activity. Storage of citral may result in the oxidation of neral and geranial to produce neric acid and geranic acid. GC/MS results showed that neral, geranial, neric acid and geranic acid were detected, while the related compounds, nerol, geraniol, citronellal, citronellol and citronellic acid were not detected either for citral stored at 5°C or at room temperature. At room temperature, geranial and neral content declined more quickly than at 5°C. The effect of citral on the incidence of disease on fruit was studied by applying citral as a fumigant. Wounded oranges inoculated with spore suspension (10⁶ spores mL⁻¹) of the fungi were placed in 5-litre plastic boxes, fumigated with 2, 6, or 15% citral, and incubated at 5°C or room temperature. Fumigation of oranges with citral in this closed system delayed the onset of sour rot at room temperature by 7 – 10 days and at 5°C, by 13 – 30 days, suggesting that volatile citral controlled G. citri-aurantii on fruit as well as in vitro. The effects of fumigation with citral on green and blue mould were more variable. Fumigation delayed the onset of green mould and blue mould at 5°C by 2 days at the higher concentrations (6 and 15%) tested, while at room temperature, spoilage was not delayed even at the highest concentration tested. Measurement of citral in the headspace of boxes containing fruit and citral-soaked pads showed that the concentration above the fruit was higher than that measured below the fruit both at 5°C and at room temperature. Phytotoxicity symptoms were observed on the upper surface of some fruit that was close to or in direct contact with the citral-soaked pad at concentrations of 6% and 15%, suggesting that phytotoxicity may have been associated with high volatile citral concentration. However, citral residue was not detected in oranges irrespective of treatment with citral, which suggested that little citral had penetrated into the peel. During storage the citral content decreased due to oxidation of geranial and neral to produce geranic acid and neric acid both at 5°C and room temperature. This may have had an impact on the efficacy of citral against the pathogens.Findings may contribute to a better understanding of the efficacy of citral when applied to the pathogens in vitro and to the development of effective control methods when applied on fruit. The possibility of combining citral treatment with other commonly used practices is also worthy of consideration. For example, citral could be combined with heat treatment to increase the volatility of the citral. In addition, incorporation of citral in a wax formulation may allow a low concentration of citral to be used in direct contact with the pathogens on fruit. Fumigation of fruit with citral may offer potential as a means to control development of sour rot of oranges, and its effects on fruit quality, flavour and nutritional aspects require further investigation.Item Open Access Characterisation of proteinaceous toxins isolated from Pyrenophora teres f. teres.(2013) Ismail, Ismail Ahmed; Able, Amanda Jane; Godfrey, Dale; School of Agriculture, Food and WinePyrenophora teres f. teres (Ptt) causes net form net blotch disease (NFNB), an important disease of barley in Australia and worldwide. This fungus uses proteinaceous toxins to cause necrosis and different isolates of Ptt differ in their ability to cause symptoms on different cultivars of barley. However, little is known about the roles of pathogen growth and individual toxins in symptom development. This project therefore aimed to determine whether there is a relationship between toxin production, fungal growth and virulence in NFNB. Conidial germination, extent of fungal growth and culture filtrate toxicity were compared for six South Australian Ptt isolates with different virulence on the barley cultivar ‘Sloop’. In addition, Ptt toxin production was optimised before identification and selection of virulence-related candidate proteins (VRCPs) for further characterisation. The biological activity of recombinant VRCPs on susceptible and resistant cultivars and VRCPs gene expression during the interaction of Sloop with each isolate were also compared. In general, the more virulent isolates had higher rates of conidial germination (both in vitro and in planta) and fungal development in planta, represented by longer hyphae and more appressoria, compared with less virulent isolates. Similarly, PttGAPDH and its transcript were more abundant during the interaction of barley with more virulent isolates. A proteomics approach was used to identify proteins unique to the more virulent isolate, proteins from bioactive fractions on either susceptible (Sloop) or resistant cultivars (CI9214 and Beecher) and proteins from the intercellular washing fluids (ICWFs) of infected barley. These analyses revealed that Ptt produced proteins between 37 and 150 kDa that have biological activity. Liquid Chromatography-Electrospray Ionisation Ion-Trap Mass Spectrometry (LC-eSI-IT MS), of individual biologically active proteins was used to identify peptides which matched to 17 proteins that belong to three groups of fungal proteins including virulence-related proteins; fungal growth and development proteins; and those with unknown function (hypothetical proteins). However, Ptt toxins were not detected in the ICWF protein profiles suggesting that Ptt toxins were either in trace amounts or might be internalised into the cell. The four VRCPs selected, were identified as hypothetical proteins with unknown function in the Ptt database. Further bioinformatic analysis characterised these VRCPs as an isochorismatase (PttCHFP1), an endo-1, 4-β- xylanase A (PttXyn11A), a glycophosphatidylinositol (GPI)-anchored common in fungal extracellular membrane (CFEM) domain-containing protein (PttGPICFEM) and an unknown proteinaceous secreted (but conserved) hypothetical protein (PttSP1). These VRCPs were heterologously expressed and characterised using gene expression studies. PttXyn11A had strong homology with the well characterised endoxylanases, TrXyn11A from Trichoderma reesei and BcXyn11A from Botrytis cinerea, known to contribute to virulence. A necrosis-inducing region on the surface of the enzyme was also identified in PttXyn11A, suggesting a potential role in necrosis induction. The culture filtrates for more virulent isolates had significantly greater xylanase activity than those from less virulent isolates. Even though heterologously expressed PttXyn11A was toxic to Escherichia coli, xylanase activity was detectable at very low levels and was not enough to cause symptoms in the bioassay. In addition, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and RT-quantitative PCR (RT-qPCR) analysis demonstrated that PttXyn11A was expressed more abundantly by the more virulent isolates compared with the other isolates in culture and during the plant-pathogen interaction. Together, these results suggest that PttXyn11A plays a role in virulence, either through its ability to degrade the plant cell wall to assist fungal growth or through its necrosisinducing ability. PttCHFP1 showed homology to an isochorismatase, an enzyme that has been proposed to have a role in plant defence via inhibition of salicylic acid production. PttSP1 showed homology to a membrane lipoprotein proposed to have a role in fungal development. Bioassay of recombinant PttCHFP1 and PttSP1 induced chlorosis symptoms in the susceptible barley cultivar (Sloop). The cysteine-rich CFEM domain identified in PttGPI-CFEM has been suggested to have an important role in hyphal attachment and fungal networking. However, E. coli was not able to express this gene probably due to its attachment to the plasma membrane and/or cell wall. Analysis of the gene expression profiles for PttCHFP1, PttGPI-CFEM and PttSP1 showed no significant differences between isolates in vitro and in planta suggesting that all isolates regulated the expression of these genes to the essential level possibly required for pathogenesis. This is the first study to identify the relationship between fungal growth and proteinaceous toxin production, characterise individual proteinaceous toxins in the mixture of Ptt culture filtrate and investigate the expression profiles of genes encoding VRCPs during the Ptt-barley interaction. This study therefore provides a better understanding of the Ptt-barley interaction by identifying the potential toxins which might lead to identify the toxin targets and ultimately support the breeding of resistant cultivars of barley.Item Open Access Effect of calcium and boron nutrition on grey mould of capsicum (Capsicum annuum L.) and fruit quality.(2014) Le, Thong Duc; Able, Amanda Jane; Scott, Eileen Sandra; McDonald, Glenn Keith; School of Agriculture, Food and WineCapsicum (Capsicum annuum L.) is mostly cultivated in humid and warm conditions, which increases disease development, particularly grey mould caused by Botrytis cinerea. Infection of capsicum fruit by B. cinerea often occurs preharvest but symptoms of grey mould are not usually visible until after harvest making the pathogen difficult to control. Appropriate fertilisation that ensures calcium (Ca) and boron (B) is sufficient in plant tissues, especially in fruit, has been suggested as an alternative to fungicides for disease management. This research studied the infection pathway of B. cinerea and the effect of Ca and B on grey mould development and quality of fruit in two capsicum cultivars (cv. Aries and cv. Papri Queen). Botrytis cinerea infected capsicum preharvest and flowers often died when inoculated at anthesis. The number of dead flowers increased when inoculum concentration increased. The extent of grey mould development on fruit inoculated preharvest was not affected by timing of inoculation [at anthesis, 3 days after anthesis (DAA) or 6 DAA], but was dependent on inoculum concentration and cultivar. When capsicum fruit were inoculated after harvest, grey mould developed most rapidly in red (R) fruit from cv. Aries and breaker red (BR) fruit from cv. Papri Queen. An inoculation of 10⁶ conidia mL¯¹ caused more disease on fruit than 10⁴ or 10⁵ conidia mL¯¹. Cv. Aries was more susceptible to B. cinerea than cv. Papri Queen regardless of whether inoculation occurred before or after harvest. The effect of both soil and foliar application of boron (B), at different concentrations, on grey mould development and fruit quality of capsicum was examined. Preharvest B application, from transplanting to harvest when fruit were mature and red, using 0.05 or 0.1 mM H₃BO₃ via soil amendment or 2.0 or 7.0 mM H₃BO₃ as a foliar spray increased B concentration in leaves and fruit of both cultivars. However, soil application was more effective than foliar application in increasing B concentration in plant tissues. Foliar application of B at low concentrations (0.025 or 0.075 mM H₃BO₃) did not increase B concentration in plant tissue. Increasing B concentration in leaf and fruit tissue reduced grey mould development on fruit inoculated with B. cinerea preharvest compared to the control, but did not affect grey mould development on red fruit inoculated with B. cinerea postharvest. Preharvest soil application of B increased shelf life of fruit, but did not affect quality of fruit including water content, firmness, total soluble solid content (TSSC) and titratable acidity (TA) at harvest or during storage. Symptoms of B toxicity were observed on leaves from plants that received high B concentration (0.1 mM H₃BO₃) in the soil, but no effect was observed on fruit. Preharvest application of calcium (Ca) via soil amendment [1.5, 4.0 or 8.0 mM Ca(NO₃)₂] or as a foliar spray [0.5 or 1.0 % w/v mM Ca(NO₃)₂] increased Ca concentration in leaves, but did not increase Ca concentration in fruit, regardless of cultivar. Soil Ca application appeared to increase Ca concentration in leaf tissue more effectively than the Ca foliar spray. Ca concentration in leaf tissue from cv. Aries was significantly higher than in leaf tissue from cv. Papri Queen when plants received the same amount of Ca, regardless of application method. Ca treatment did not affect quality of fruit at harvest or during storage. Preharvest application of Ca reduced grey mould development on fruit that had been inoculated with B. cinerea preharvest, but did not reduce grey mould in fruit inoculated postharvest. Symptoms of Ca deficiency were observed on plants that received no Ca or low Ca concentration [1.5 mM Ca(NO₃)₂] from transplant to fruiting. Dipping and vacuum infiltration with calcium chloride (CaCl₂.2H₂O) did not increase Ca concentration in flesh after treatment, but vacuum infiltration did increase Ca concentration in flesh after 10 days of cool storage (10°C). Ca treatment after harvest did reduce grey mould development on fruit, but did not affect the quality of fruit during storage. A directly inhibitory effect of Ca on fungal growth was responsible for reducing grey mould development on fruit. In conclusion, capsicum was most sensitive to infection by B. cinerea at anthesis and high inoculum concentrations caused a greater disease incidence in capsicum fruit, regardless of whether inoculation occurred preharvest or after harvest. Reducing inoculum concentration, especially during flowering, is therefore recommended to reduce losses in capsicum. Preharvest application of Ca or B may be used as an alternative method to reduce grey mould on capsicum fruit, but they had no effect on fruit quality. Postharvest application of Ca could also be recommended for cv. Aries fruit before or during storage for controlling grey mould on fruit. Findings in this research may therefore provide basic knowledge for management of B. cinerea in the capsicum industry.Item Open Access Effect of nutrition on postharvest quality and grey mould development in strawberries.(2008) Naradisorn, Matchima; Able, Amanda Jane; Scott, Eileen Sandra; Sedgley, Margaret; School of Agriculture, Food and Wine : Plant and Food ScienceStrawberries are an extremely perishable fruit mainly due to their soft texture and sensitivity to fungal infection. The fungal pathogen Botrytis cinerea is responsible for grey mould on strawberries and is the main causal agent of postharvest decay and subsequent economic loss. As an alternative to fungicides, manipulation of plant nutrition, such as calcium and boron, has been suggested as a means of disease management. This project investigated the effects of calcium and boron application on fruit quality and grey mould development in strawberry. The effect of calcium on fruit quality, grey mould development and leaf blight in strawberry cultivars ‘Aromas’ and ‘Selva’ was investigated through preharvest and postharvest applications. To determine the effect of preharvest application, calcium sulphate in 0.25X strength Hoagland’s solution was applied at 0, 100, 300 and 500 ppm Ca through fertigation. Fully-ripened fruit were harvested and evaluated for postharvest quality at harvest and then after storage at 10⁰C, 90±5% RH for 2 to 10 days. Although fruit firmness of both cultivars declined slightly during storage, this was not affected by preharvest calcium application. Similarly, preharvest calcium treatment had no effect on the external appearance, pH, soluble solids content (SSC) or titratable acidity (TA). No grey mould development was observed on fruit at harvest when flowers were inoculated with a conidia suspension of B. cinerea (10⁴ conidia per mL). However, fruit harvested from plants that received calcium at any concentration had less incidence of grey mould during storage at 10⁰C, 90±5% RH for 14 days than fruit harvested from plants that received no calcium for both cultivars. For ‘Aromas’, 79% and 51% of fruit, and for ‘Selva’, 69% and 43% of fruit, showed rot when treated with 0 and 500 ppm Ca, respectively. The shelf life of ‘Aromas’ and ‘Selva’ increased by about 8% when plants received 500 ppm Ca in comparison with plants that received 0 ppm Ca. After 7 days of incubation at 22 to 24⁰C, there was no difference between blight lesions on wound-inoculated detached leaves from different calcium treatments for either cultivar. However, the lesions on ‘Selva’ were smaller than on ‘Aromas’. The calcium levels in leaves from plants that received calcium at any concentration were adequate for strawberry growing and significantly higher (P < 0.05) than in leaves from plants that received 0 ppm Ca. However, calcium treatment did not ensure transfer of calcium to fruit tissues. Calcium lactate and calcium chloride were used as postharvest calcium treatments at 1500, 3000 and 4500 ppm Ca. Fruit of ‘Selva’ were dipped in calcium solution for 5 min and wound-inoculated with B. cinerea (10⁶ conidia per mL). Calcium lactate and calcium chloride at 3000 and 4500 ppm Ca, respectively, were most effective in delaying Botrytis rot development on ‘Selva’ after 7 days of storage at 10⁰C, 90±5% RH. Storage for least 24 h after calcium dips prior to inoculation was required to delay the development of fruit rot. Fruit harvested early in the season seemed to be less susceptible to grey mould than those harvested later. However, calcium treatment tended to be more effective when applied to late-season fruit. Preharvest boron treatment, applied as for calcium but at 0, 0.25, 0.5 and 1.0 ppm B, had no effect on fruit firmness of either cultivar. However, firmness of ‘Aromas’ fruit was slightly greater than ‘Selva’ fruit for all treatments. The amount of boron applied had no effect on the external appearance, pH, SSC or TA for either cultivar after storage of fruit for up to 10 days. Application of boron had no effect on fruit grey mould development in either cultivar. Furthermore, boron had minimal effect on the incidence of blight on woundinoculated detached leaves of ‘Aromas’ 7 days after inoculation. However, blight lesion diameters on ‘Selva’ leaves in the 1.0 ppm B treatment (8.0 mm) were significantly smaller (P < 0.001) than in the 0 ppm B treatment (13.0 mm). Phytotoxicity was observed in boron treatments even at the level considered optimum for strawberry growing. Severity increased with increasing boron concentration but no consistent effect on flower death or flower abortion was observed. In conclusion, strawberry is sensitive to boron toxicity. Calcium may enhance fruit firmness and, consequently, delay grey mould development if calcium penetrates the fruit. Postharvest calcium treatment tended to be more effective in delaying development of grey mould when applied to late-season fruit. Calcium lactate is a potential alternative to calcium chloride for reducing decay caused by B. cinerea in strawberry without providing undesirable bitterness. This finding may provide a basis for application in industry.Item Open Access The genetic basis of barley black point formation.(2008) March, Timothy; Able, Amanda Jane; Able, Jason Alan; Schultz, Carolyn Jane; School of Agriculture, Food and Wine : Plant and Food ScienceBlack point of barley grain refers to a discolouration of the embryo end of the grain. Historically black point has been proposed to be due to fungal colonisation of the grain. However, Koch’s postulates have yet to be satisfied. The discolouration occurs during grain fill in response to high humidity or rainfall during the grain filling period. In wheat, which is also affected by black point, the discolouration has been proposed to be due to the oxidation of phenolic acids within the grain to form discoloured end products. Within this study, two approaches were investigated in order to understand the proteins and genes associated with this disorder. Firstly, a proteomics approach enabled the identification of individual proteins associated with black point. Two-dimensional gel electrophoresis was used to compare the proteome of the husk and whole grain tissue of mature black pointed and healthy grain. Very little watersoluble protein was extracted from the husk tissue. However, a significantly larger amount of protein was extracted using a salt extraction buffer, indicating the husk proteins were mostly cell wall bound. Due to the effect of residual salt and low protein concentrations these proteins were not conducive to analysis using two-dimensional gel electrophoresis. Further experiments using acid hydrolysis of the husk tissue and subsequent amino acid analysis revealed that the proteins were bound to the husk cell walls via covalent bonds. In contrast, large quantities of protein were obtained from the whole grain samples. This allowed statistically significant comparisons to be made between gels from healthy and black pointed grains. Two proteins were identified as being more abundant in black pointed grains. Mass spectrometry identified these as isoforms of barley grain peroxidase 1 (BP1). In addition, three proteins were identified as being more abundant in healthy grain. Mass spectrometry revealed these to be isoforms of the same protein with sequence similarity to a partial EST sequence from barley. Using 3' RACE the entire coding sequence of the gene was isolated which revealed that it encoded a novel putative late embryogenesis abundant (LEA) protein. Northern blot analysis was performed for BP1 and LEA and showed that gene expression differences could not account for the differences seen in protein quantities. Western blot analysis revealed that the LEA protein was biotinylated in vivo which is consistent with similar LEA proteins from other plant species. To further understand the role of these proteins in black point, antibodies were raised against the two proteins. Subsequent immunolocalisation studies indicated BP1 was present throughout all tissues of the grain whilst LEA was most abundant in the embryo and aleurone tissue. The second major area of investigation within this thesis was to further delineate the previously identified quantitative trait loci (QTL) associated with black point in barley. Previous studies have reported QTL for black point and kernel discolouration in both barley and wheat. Comparison of the published QTL revealed a locus on the short arm of chromosome 2H to be of particular interest. To identify genes underlying this QTL the genomes of barley, wheat and rice were compared. An in silico approach showed that the QTL shared macro-synteny with rice chromosomes 4 and 7. From the rice genome sequence, barley ESTs with sequence similarity were selected. In total, 20 ESTs were selected based on two main criteria: their putative role in black point and also being evenly spread across the region of the QTL length. These QTL were mapped within the Alexis x Sloop double haploid population. This approach revealed that there was some conservation of synteny but also identified clear boundaries where synteny between barley and rice had been lost since divergence. Significantly, the additional markers mapped to this region have enabled the initial black point QTL to be reduced from approximately 30cM to 20cM. In conclusion, this study has added significant knowledge our understanding of the genetics of black point in barley through the use of two approaches. The proteomics approach has aided in understanding the biochemical processes occurring within the grain in response to black point. The comparative genetics approach has aided in understanding the genetic control of an important region of the genome influencing black point susceptibility. Combined, these findings will direct future research endeavours aimed at producing black point resistant barley cultivars.Item Open Access An investigation of bread wheat meiosis via proteomics and gene-targeted approaches: the isolation and characterisation of four meiotic proteins.(2011) Khoo, Kelvin Han Ping; Able, Jason Alan; Able, Amanda Jane; School of Agriculture, Food and WineDuring the early stages of meiosis, three key processes occur: chromosome pairing, synapsis and DNA recombination. Chromosomes are first replicated during interphase, after which they are aligned together in a non-random fashion to enable the installation of the synaptonemal complex (SC) along the chromosome axes leading to synapsis. Recombination machinery then enables strand invasion to occur, which then leads to the formation of chiasmata and ultimately, genetic recombination. Meiosis is further complicated in organisms with multiple genomes such as allohexaploid bread wheat (Triticum aestivum L.) which has three genomes (inherited from similar yet distinct progenitors), each with seven chromosomes. Thus a large number of proteins are likely to be required for the successful execution of this biological process. The first approach in this study used proteomics to identify proteins that have possible roles during the early stages of wheat meiosis. Total protein samples isolated from staged meiocytes (specifically from pooled stages of pre-meiotic interphase to pachytene and from telophase I to telophase II) of wild-type Chinese Spring and the Pairing homoeologous deletion mutants, ph1b and ph2a, were analysed by 2-dimensional gel electrophoresis (2DGE). This resulted in identifying six differentially expressed protein spots (designated KK01 to KK06); from which three full-length coding sequences and one partial coding sequence of the candidate genes encoding these proteins were isolated (a putative speckle-type POZ protein, a pollen-specific SF21-like protein, a putative HSP70-like protein, as well as a partial hexose transporter peptide). Southern blot analysis revealed that these genes were spread across four different chromosome groups (2, 7, 5 and 1 respectively) with a copy on each of the three genomes (A, B and D). Q-PCR analysis of these four genes across the two pooled meiotic stages and various genotypes suggests that both KK01 and KK06 have roles during the early stages of meiosis and that they may be directly/indirectly regulated by a combination of elements within the Ph1 and Ph2 loci. The high level of KK03 mRNA transcript detected in the later stages of meiosis is consistent with its role as a pollen-specific protein-encoding gene. In contrast, KK04 expression suggests that it is post-transcriptionally regulated resulting in KK04 being translated in the ph2a mutant. Both the speckle-type POZ protein and putative dnaK/HSP70 protein were also shown to interact with DNA in vitro. The second approach of this study focused on isolating and characterising wheat homologues of two known meiotic proteins, namely PHS1 and ZYP1. In the maize PHS1 mutant Zmphs1-0, homologous chromosome pairing and synapsis are significantly affected, with homoeologous chromosome interactions occurring between multiple partners. More recently, co-immunolocalisation assays using anti-PHS1 and anti-RAD50 antibodies showed that both proteins had similar localisation patterns in the wild-type maize plants and that RAD50 localisation into the nucleus was affected by the absence of PHS1 thus implicating PHS1 as a regulator of RAD50 nuclear transport. In this study, the full-length coding transcript of wheat PHS1 (TaPHS1) was isolated, sequenced and characterised. TaPHS1 is located on chromosome group 7 with copies on the A, B and D genomes. Expression profiling of TaPHS1 in both wild-type and the ph1b mutant during and post-meiosis show elevated levels of TaPHS1 expression in the ph1b background. The TaPHS1 protein has sequence similarity to other plant PHS1/PHS1-like proteins but also possesses a unique region of oligopeptide repeat units. DNA-binding assays using both full-length and partial peptides of TaPHS1 show conclusively that TaPHS1 is able to interact with both single- and double-stranded DNA in vitro, even though no known conserved DNA-binding domain was identified within the TaPHS1 sequence, indicating TaPHS1 possesses a novel uncharacterised DNA-binding domain. Immunolocalisation data from assays conducted using an antibody raised against TaPHS1 demonstrates that TaPHS1 associates with chromatin during early meiosis, with the signal persisting beyond chromosome synapsis. Furthermore, TaPHS1 does not appear to co-localise with the asynapsis protein – TaASY1 – possibly suggesting that these proteins are independently coordinated. Combined, these results provide new insight into the potential functions of PHS1 during early meiosis in bread wheat. Similar to PHS1, Arabidopsis knock-down mutants of ZYP1 also display non-homologous chromosome interactions. ZYP1 has previously been characterised as a SC protein required for holding homologous chromosomes together in other species. In this study, the full-length coding sequence of the wheat ZYP1 (TaZYP1) homologue was isolated, sequenced and characterised. Expression of TaZYP1 analysed by Q-PCR across wild-type, ph1b and multiple Taasy1 mutants during meiosis showed an approximate 1.3-fold increase in the ph1b mutant. In addition, DNA-binding assays demonstrate that TaZYP1 interacts with dsDNA under in vitro conditions while immunolocalisation (using an anti-TaZYP1 antibody) across wild-type, ph1b and Taasy1 revealed the spatial and temporal localisation pattern of TaZYP1. Taken together, these results show that TaZYP1 plays an identical role to its homologues in other species as a SC protein and is affected by reduced levels of TaASY1 in wheat. This body of work utilised a two-pronged approach to investigate meiosis in wheat with the overall outcome of identifying new meiotic proteins as well as characterising the wheat equivalents of two known meiotic proteins previously reported in other organisms. To this end, two previously uncharacterised wheat proteins with possible roles (involving interactions with chromatin) during meiosis have been successfully identified using the proteomics approach while both TaPHS1 and TaZYP1 have been characterised with antibodies raised against both these proteins. The characterisation of TaPHS1 and its DNA-binding capabilities, both in vitro and in planta, has shed light on a previously unknown function of the PHS1 protein while the localisation profile of TaZYP1 in Taasy1 mutant lines has contributed to our understanding of how ASY1 levels can affect chromosome pairing in wheat.Item Open Access Regulation of candidate genes in black point formation in barley.(2012) Walker, K. Ryan; Able, Amanda Jane; Able, Jason Alan; Mather, Diane Elizabeth; School of Agriculture, Food and WineBlack point of barley refers to discolouration of the embryo end of the grain. Downgrading of malting barley to feed grade due to black point results in significant economic loss to the Australian barley industry. Given that black point normally occurs in regions of Australia that experience high humidity during grain fill, humidity most probably contributes to the severity of black point in susceptible varieties. Previous studies have excluded fungal infection as a cause but enzymatic browning reaction has been recently hypothesised as responsible for black point. More specifically, a role for peroxidases has been proposed. The first major focus of this study was to confirm under what environmental conditions black point formation was likely to occur and whether there was genetic variation contributing to the phenotype. The occurrence of high humidity and low temperatures was associated with the formation of black point in susceptible varieties, with early maturing varieties being more susceptible to black point. These environmental conditions probably create a moist environment during grain development in which the developing grain cannot dry out, enabling stress or wounding to the embryo that subsequently results in black point formation. Analysis combining two South Australian sites (Hatherleigh and Port Wakefield, SA) identified QTL for black point formation on chromosomes 2H (QBpt.AlSl-2H) and 3H QBpt.AlSl-3H) at positions 83.4 cM and 102.6 cM respectively. Additive by environment effects were substantial at both QTL. Linkage of the QTL on chromosome 2H with the earliness per se (eps2) locus and the observation that early maturing varieties were usually more susceptible to black point established a probable association between earliness and black point susceptibility. When an early maturing(susceptible) variety was planted later so that it matured at the same time as a later maturing (tolerant) variety there was no significant difference in black point scores. The second focus of this study was to characterise a number of candidate genes more than likely linked to black point by investigating expression levels during grain fill and subsequently mapping the genomic regions responsible for those changes in expression. Candidate genes chosen were Quinone Reductase (HvQR), Phenylalanine Ammonia Lyase (HvPAL), Barley Peroxidase 1 (HvBP1), stress-related Peroxidase (HvPrx7) and Lipoxygenase A (HvLoxA). Differential expression as detected using northern analysis, between susceptible and tolerant varieties, was only observed for HvBP1, HvPrx7 and HvQR. Quantitative PCR (qPCR)confirmed that HvBP1 and HvPrx7 expression was up to two times higher in black point susceptible varieties during all stages of grain development, while HvQR expression was significantly higher in the hard dough and mature stages of grain fill in susceptible varieties. Increased expression for HvBP1 and HvPrx7 (approximately two-fold) was also apparent in the tolerant variety Alexis between symptomatic and asymptomatic grains. The qPCR data was then used as a quantitative trait, to score the expression of these candidate genes in an Alexis/Sloop double haploid (DH) mapping population. Areas of the genome potentially involved in the regulation of these candidates (expression QTL or eQTL) were mapped on chromosomes 2H (for HvPrx7 and HvBP1) and 5H (for HvQR and HvBP1). The eQTL for HvPrx7 and HvQR were located in the same regions as the corresponding genes, suggesting their expression is regulated via cis-acting factors. In contrast, while HvBP1 is located on 3H, eQTL were located on 2H and 5H suggesting trans-acting factors were involved. The use of comparative mapping studies between barley and rice identified a number of transcription factor genes within these eQTL. The final component of this study was to investigate how HvBP1 and HvPrx7 expression might be affected by examining their promoters and potential interactors with those promoters. Promoter regions for the susceptible variety Sloop and tolerant variety Alexis were isolated, compared and analysed for known motifs. Particular emphasis was placed on those elements that were associated with embryo and endosperm specific expression or responses to environmental stresses. Several regions containing single nucleotide polymorphisms (SNPs) between the promoters from the tolerant and susceptible varieties were identified. A 160 bp region for HvBP1 and 380 bp region for HvPrx7 were used in Yeast One Hybrid (Y1H) screening to identify potential regulatory proteins. In particular, a potential bZIP-containing factor which interacted with the promoter of HvPrx7 was further characterised. Interaction was confirmed by a gel shift assay and gene expression by northern analysis showed expression at the milk, soft dough and hard dough stages of grain development. Increased expression was apparent in the susceptible variety Sloop. The eQTL, Y1H and environmental studies have furthered our understanding of genes that could be involved in the regulation of black point formation under conditions of low temperature and high humidity. This information will contribute to assessing the roles these genes play in black point formation under certain environmental conditions, and more broadly, will assist in improving breeding for resistant barley varieties.Item Open Access Ripening behaviour of capsicum (Capsicum annuum L.) fruit.(2007) Pham Thi, Ngoc Thang.; Able, Amanda Jane; Sedgley, Margaret; School of Agriculture, Food and Wine : Plant and Food ScienceFruit of Capsicum annuum L. (capsicum or pepper) are one of the major sources of red food colourant and pungency for spice production. In the spice production industry, fruit are mechanically harvested at different ripeness stages and fruit colour needs to be synchronised before being processed. However, even though capsicum ripens normally on the plant it often fails to ripen fully and turn red once harvested at the green stage. Attempts to promote ripening of harvested fruits have had limited success and the reason for this has been unclear. This project, therefore, investigated ripening behaviour on and off the plant of capsicum fruit grown in Australia and examined effects of pre- and postharvest applications on ripening of green harvested fruit. To examine ripening behaviour on and off the plant, capsicum fruit from three different cultivars (a mild paprika type cv. “Papri Queen”, a cayenne chilli cv. “Caysan”, and a sweet type bell pepper cv. “Aries”) were either allowed to ripen naturally on the plant or harvested at three different maturity stages: light green, deep green and breaker. Harvested fruit were stored individually at room temperature and several ripening characteristics including internal ethylene (C2H4) and carbon dioxide (CO2) concentration, extractable colour, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and oxidase activity, and total soluble solid content (TSSC) were studied during storage. There was very limited involvement of C2H4 during ripening of capsicum and the change in ACC synthase and ACC oxidase (two enzymes in C2H4 biosynthesis pathway) activity was not closely related to that of C2H4. However, it appeared that colour development in cv. “Papri Queen” was closely associated with what C2H4 production did occur while a climacteric-like peak of C2H4 could be observed in all fruit from cv. “Caysan”. For all three cultivars, the level of internal CO2 concentration, extractable colour and TSSC were greater in fruit ripened on the plant followed by fruit harvested at the breaker, deep green and light green stage, respectively. Fruit harvested at the light green stage failed to change colour properly and had very low levels of internal CO2 concentration and TSSC while fruit harvested from the breaker stage onwards ripened normally and developed sufficient colour for spice processing. This may suggest a role of external carbon-supply during ripening. To study the effect of the external-carbon supply during ripening, the stem of fruit were cinctured when fruit reached the light green stage and fruit were left to ripen on the plant. Cincturing delayed colour development of fruit by approximately five days but cinctured fruit were still able to turn red and develop extractable colour higher than the acceptable level of 140 ASTA units. Cincturing did not significantly alter other ripening behaviour such as CO2 concentration or TSSC. The lack of external carbon-supply is, therefore, unlikely to play a major role in the failure of green harvested fruit to ripen. To study the effect of application of plant growth regulators (both pre- and postharvest), an effective method of solution application utilising cincturing was firstly developed. Different plant growth regulator solutions including ethephon, naphthalene acetic acid, abscisic acid, jasmonic acid, sucrose, and different combinations of these were applied to fruit at the light green stage to study preharvest effects on ripening parameters during storage. Only treatment with high concentrations of ethephon increased the extractable colour higher than the acceptable level of 140 ASTA units and induced the complete degradation of chlorophyll. To study effects of postharvest application, 10 µL of various plant growth regulators was dropped into the hole created on the stem of harvested fruit for ten consecutive days. Treatment with ethephon significantly increased extractable colour and degraded chlorophyll content of fruit. Pre- and postharvest ethephon treatment strongly up-regulated Capsanthin-capsorubin synthase (Ccs) gene expression in a manner similar to the up-regulation of Ccs observed in fruit ripened on the plant. This explains the effect of C2H4 on colour development and also indicates the possible reason for the failure of green harvested fruit to ripen. However, the Ccs gene expression and chlorophyll degradation induced by ethephon was not visible until 14 days after harvest which indicated it may not be a direct effect and other signal transduction factors may be involved. When fruit are ripened on the plant, colour development may, therefore, be induced by ripening-related factors (other than C2H4) which is possibly inhibited or inactivated when fruit are harvested at the green stage. C2H4 application to fruit at this stage may help to reactivate or recover these factors which in turn induce colour development. Thus, although capsicum fruit show typical non-climacteric behaviour, C2H4 appears to be involved in some aspects of the ripening process.Item Open Access Role of Pyrenophora teres toxins in net blotch of barley.(2007) Sarpeleh, Abolfazi; Able, Amanda Jane; Wallwork, Hugh; Catcheside, David Edward Arnold; School of Agriculture, Food and Wine : Plant and Food SciencePyrenophora teres, the causal agent of net blotch of barley (Hordeum vulgare L.), exists in two forms; P. teres f. teres and P. teres f. maculata. Both forms induce a combination of brown necrotic spots and extensive chlorosis in susceptible barley cultivars. Although a number of low molecular weight compounds (LMWCs) have been previously isolated from P. teres culture filtrates, they only induced certain components of symptoms. Fungal metabolites were extracted from culture filtrates of both forms of the pathogen and separated into low (<3kDa) and high molecular weight compounds (HMWCs, >10 kDa) with each fraction inducing a component of the net blotch symptoms in a barley leaf toxicity assay. Inactivation of both LMWCs (<1kDa) and HMWCs resulted in loss of activity confirming their potential role in symptom development. Low molecular weight compounds induced chlorosis and water soaking but not the brown necrotic spots or lesions usually seen during the infection of barley by P. teres. The high molecular weight compounds (>10 kDa) induced the brown necrotic spots or lesions with no chlorosis evident. Further characterisation of LMWCs showed that they are not host specific while HMWCs exhibited host specificity. LMWCs were purified and further analysed using high voltage paper electrophoresis, staining and mass spectrometry. Electrophoretic properties and staining of the LMWCs with ninhydrin indicated that both forms of P. teres produced similar LMWCs in the conditions grown. Each form produced eight ninhydrin-positive compounds with the samerelative mobilities. Each individual compound was shown to induce chlorosis in excised barley leaves. All compounds except the one isolated in this study appear to be derivatives of or are the previously described compounds; N-(2-amino-2carboxyethyl) aspartic acid (Toxin A), aspergillomarasmine A, anhydroaspergillomarasmine A and aspergillomarasmine B. The exception is a bioactive UV absorbing LMWC which appears to be a reductive conjugation of the α-keto acid of phenylalanine with Toxin A. The HMWCs (>10kDa) were proteinaceous since they were identifiable using Coomassie staining. Additionally, the loss of activity that occurred with incubation at 40, 60, and 80 °C for 30 and 60 min followed a pattern fairly typical for protein denaturation. Further, treatment with protease decreased their phytotoxicity in proportion to the amount of enzyme used. Enzyme and heat treatment of proteins extracted from each form showed that proteins of P. teres f. teres are more resistant to heat and enzyme treatment compared with those of P. teres f. maculata. This suggests the protein(s) involved in symptom induction by P. teres f. teres and P. teres f. maculata are different which contributes to the difference in the symptom expression during the interaction between the pathogens and barley. Proteinaceous metabolites extracted from P. teres f. teres and P. teres f. maculata ranged from 10 to 100 kDa. Fractions purified using gel filtration had biological activity when they contained eight proteins when extracted from P. teres f. maculata (90, 80, 75, 55, 48, 35, 14 and 12 kDa) and six proteins when extracted from P. teres f. teres (90, 80, 55, 48, 14 and 12 kDa). Additionally, intercellular washing fluids (IWF) extracted from barley plants inoculated with both forms of P. teres, contained proteins of the same size as those in the biologically active fractions extracted from culture filtrates of P. teres f. maculata (80, 14 and 12 kDa) and P. teres f. teres (80, 48 and 14 kDa). Automated MS/MS sequencing of the biologically active proteins showed no resemblance to the sequences or conserved domain information available in public databases and as a consequence, these proteins were considered as novel proteins for P. teres. However, exact short matches with fragments resulting from the 80, 48 and 14 kDa proteins, showed considerable homology with ATP-binding cassette (ABC) transporters and their components, cellulases, serine proteinases as well as some hypothetical proteins isolated from different fungal species. Reaction of six plant species including one susceptible barley cultivar (Sloop) and one resistant line (CI9214) to P. teres showed that partially purified proteins induce the symptoms selectively in barley cultivars where the proteinaceous metabolites only induced brown necrotic spot/lesions in barley with a greater response seen on the susceptible cultivar Sloop when compared to the resistant line CI9214. No symptoms were seen on other plant species employed in this study suggesting that the proteinaceous metabolites isolated in this study are host specific phytotoxins. This research has allowed the first isolation of proteinaceous host-specific toxins from P. teres as well as the identification of a UV-sensitive LMWC phytotoxin not previously described. Proteinaceous toxins induced brown necrotic spots/lesions specific to the host while the LMWCs induced chlorosis in a number of different plant species. This contributes significantly to the body of knowledge defining how symptoms are caused during the pathogenicity process in the interaction between P. teres and barley.Item Open Access Strawberry powdery mildew: epidemiology and the effect of host nutrition on disease.(2007) Palmer, Sarah A.; Able, Amanda Jane; Scott, Eileen Sandra; Stangoulis, James Constantine Roy; School of Agriculture, Food and Wine : Plant and Food ScienceKnowledge of disease epidemiology and the impact of plant nutrient status on development of disease is fundamental in establishing effective management strategies for crop pathogens such as Podosphaera aphanis Br. (Braun et al., 2002), the causal agent of powdery mildew on strawberries. The following study investigated the conditions conducive for powdery mildew in strawberry crops in South Australia, the effect of foliar concentration of potassium and calcium on yield and pathogen development on the strawberry cultivars Aromas (resistant to powdery mildew) and Selva (susceptible), the potential for use of foliar-applied potassium silicate to control disease and identification of genes differentially expressed during disease. Meteorological conditions associated with establishment of powdery mildew were observed over three consecutive seasons in commercial strawberry crops grown in Woodside, South Australia. Conducive conditions appear to be >28 oC, <55 % relative humidity (RH) with no rain during the day (for conidiation), followed by a night with >10 oC, >90 % RH and no rain (for germination). Colony development was then promoted by days of >15 oC and <70 % RH, with nights of >8 oC, >80 % RH and less than 2 mm rain in every 24 hour period. These are consistent with epidemiological studies of this pathogen (Peries, 1962a; Jhooty and McKeen, 1964; Mukerji, 1968; Perera and Wheeler, 1975; Byrne et al, 2000; Miller et al, 2003; Blanco et al, 2004; Davik and Honne, 2005; Amsalem et al, 2006). This knowledge may facilitate prediction of times considered high risk for establishment of powdery mildew in strawberry crops. Subsequently, this may allow optimisation of fungicide application and improved management of this disease and reduced yield loss and management expenses. P. aphanis developed at an increased rate on leaves of Selva with low calcium content compared with development on normally fertilised leaves. Increased numbers of conidia germinated successfully on leaves of Aromas with low calcium content compared with development on normally fertilised Aromas leaves, however, the germinated conidia still failed to develop into sporulating colonies. Potassium nutrition had no obvious effect on P. aphanis development. Foliar concentration found to be adequate for growth of cultivars, Selva and Aromas were 6.0 mg/g and 4.5 mg/g Calcium, dry weight and 11.0 mg/g and 12.5 mg/g potassium, dry weight (respectively). Potassium silicate, buffered to pH 7.0 and applied as a foliar fertiliser, reduced the severity of powdery mildew below the economic threshold, though not below the disease severity on plants treated with the fungicide, Systhane® (Bayer CropScience). As potassium silicate can be produced organically this compound may provide a useful management tool for both organic and conventional strawberry growers. Although the cultivar Aromas was not immune to disease under conducive conditions and high inoculum load in the field, inoculation of healthy Aromas plants with P. aphanis in the laboratory failed to produce disease. Conidia were seldom found attached to the leaf surface of healthy Aromas leaves. Germination and subsequent colony development were also not observed in inoculated samples. This suggests there is some mechanism of Aromas that inhibits development of this fungus. Preliminary investigation of differential expression in Aromas inoculated with P. aphanis, identified sequences with homology to a putative antimicrobial protein and photosynthesis-related genes. The results of these studies should enable growers to increase both crop yields and control of powdery mildew, one of the major economic diseases in South Australia. The epidemiological knowledge attained will be valuable, and may provide the basis for future forecast modelling for P. aphanis in strawberry crops in South Australia. Recommendations for calcium and potassium leaf content will allow growers to monitor their fertiliser regime for increased yield of these cultivars. Aromas was identified as a powdery mildew resistant cultivar potentially suitable for production in South Australia, and the genes associated with this resistance response may be used in studies of Fragaria species and breeding for powdery mildew resistant cultivars.Item Open Access Susceptibility of native plant species to Phytophthora cinnamomi and the spread of Phytophthora dieback in South Australia.(2012) Kueh, Kiong Hook; Scott, Eileen Sandra; Able, Amanda Jane; Facelli, Jose Maria; Franco, Chris; School of Agriculture, Food and WinePhytophthora dieback, caused by Phytophthora cinnamomi Rands, affects a wide range of Australian native plants. In South Australia, the pathogen has affected large areas of native vegetation to threaten plant biodiversity. Lack of information on the disease in the local environment hampers management. The main objectives of this project were to: a) determine the rate of pathogen and disease spread in naturally infested native vegetation, b) assess the susceptibility of plant species native to South Australia to the disease and c) assess ability of antagonistic soil actinomycetes to protect susceptible species from Phytophthora dieback. A confirmed P. cinnamomi-infested site, with gentle slope, at Mount Bold Reservoir Catchment Reserve in the Mount Lofty Ranges, was selected to assess pathogen and disease spread in native vegetation. The soil was loamy sand. The vegetation was open woodland dominated by Eucalyptus obliqua L’Hérit with an understorey dominated by Xanthorrhoea semiplana F. Muell, a highly susceptible species which was used as an indicator to assess disease spread. An area of 70 m x 70 m, extending from two disease fronts into the adjoining healthy vegetation, was marked into 10 m x 10 m quadrats. The number of dead and dying X. semiplana was counted and soil samples from each quadrat, collected every spring and autumn from 2008 to 2010, were baited for P. cinnamomi using cotyledons of E. sieberi L.A.S. Johnson. P. cinnamomi was regularly detected along the disease front. However, the pathogen did not spread across the slope into the adjoining healthy vegetation despite annual rainfall of 626 to 900 mm for three consecutive years (2008 to 2010). The slow spread of the pathogen was reflected in the small numbers of dead and dying X. semiplana observed in each quadrat at each assessment time. The limited spread of the pathogen may be due to unfavourable weather conditions. In winter (June to August), when the precipitation was high (ca. 50% of the annual rainfall), soil temperature was generally too low (average temperature 9.3°C) for formation of sporangia. On the contrary when the temperature was warm (≥ 15°C) during spring (September to November) and autumn (March to May), the average soil water potential, ≤ -200 kPa, may have been too low for movement of zoospores. Further, sporadic distribution of P. cinnamomi and the patchiness of disease spread might have reflected the efficiency of the baiting technique. Thirty-seven South Australian native plant species, including 15 threatened or locally endangered species, were assessed for susceptibility to Phytophthora dieback in a greenhouse from October 2009 to July 2010. Seedlings or cuttings were raised in potting mix for native species then transplanted to 15 cm-diameter pots filled with limed University of California mix or Bio Gro® (Bio Gro, South Australia). Plants were inoculated with P. cinnamomi via pine wood-inoculum plugs when up to 6 months old, maintained in moist conditions and monitored for disease symptoms for 3 to 6 months. Twenty-four of the 37 species studied, including 8 threatened species, were susceptible to the disease. Nine of these 24 species were ranked as highly susceptible. Another nine species were assessed as resistant. All species classed as susceptible were trees or shrubs while herbs were unaffected. In South Australia, where native vegetation has been extensively cleared or degraded, Phytophthora dieback represents an additional threat to the remnant native flora that might cause the extinction of native plant species, particularly the rare and endangered species, if not brought under control. Actinomycetes were isolated from soil collected from roots of Acacia pycnantha Benth and young, healthy X. semiplana growing close to dead X. semiplana at the field site. Of 127 actinomycetes isolates selected, 78% inhibited P. cinnamomi in dual culture. Eight Streptomyces spp. which exhibited strong to weak antagonism, were compared in the greenhouse for ability to protect 2-month old E. sieberi. One isolate delayed infection of E. sieberi by P. cinnamomi, although none prevented disease. The high soil moisture (≥ -10 kPa) required to induce disease was probably not conducive for the growth of the actinomycetes. Knowledge generated in this project can be used in Phytophthora management to help prioritise threatened plant species in South Australia for protection, inform revegetation programs and to provide the basis for further research in the state.Item Open Access Understanding the physiological mechanisms of ripening in capsicum.(2014) Wan Kamaruddin, Wan Mohd Aizat; Able, Amanda Jane; Able, Jason Alan; Stangoulis, James Constantine Roy; School of Agriculture, Food and WineCapsicum (Capsicum annuum L.) is considered a non-climacteric fruit, exhibiting limited respiration and ethylene levels. The physiological mechanisms of ripening in capsicum have not been fully understood to date, especially the probable reason behind the non-climacteric behaviour. In this thesis, the protein (Chapter 3) and metabolite (Chapter 4) profiles of capsicum at different ripening stages have been reported. Several proteomic and metabolic candidates, for example ACC oxidase (ACO) enzyme, sugars (glucose, fructose, sucrose) and malate were chosen and analysed in different tissue types (peel, pulp and seeds/placenta) and cultivars with different ripening times (Chapter 5). The results suggested that some of these candidates were differentially present in different tissues and cultivars which implied that ripening could be regulated spatially and temporally. Furthermore, proteomic analysis also identified an ACO isoform 4 (CaACO4) which was found during capsicum ripening onset and corresponded to the increase in the overall ACO activity (Chapter 3). The expression of several ACO isoforms including CaACO4, and other identified ACC synthase (ACS) and Ethylene receptor (ETR) isoforms were therefore characterised to shed some light on their roles in this non-climacteric fruit (Chapter 6). CaACO4 was the only ACO isoform expressed significantly higher during capsicum ripening onset, confirming the earlier proteomic results. The expression of several ACS and ETR isoforms, normally associated with the climacteric increase of ethylene in tomato (a close relative of capsicum), was also limited as was ACS activity and ACC content. The production of ACC, as an ethylene precursor, may therefore be the rate-limiting step for ethylene production in non-climacteric capsicum. The postharvest application of ethylene did not promote capsicum ripening or induce the expression of most ACO, ACS and ETR isoforms, suggesting they are not regulated by ethylene as usually observed in climacteric fruit such as tomato. However, 1-methylcyclopropene treatment significantly delayed capsicum ripening postharvest, particularly when applied at Breaker stage (the onset of ripening), suggesting that blocking ethylene perception could affect ripening and that the basal level of ethylene normally produced in non-climacteric fruit may be (partially) required for ripening (Chapter 6). Other proteomic candidates such as Copper chaperone, TCP chaperone, Cysteine synthase and Spermidine synthase were also isolated and investigated due to their possible roles in capsicum ripening. However, unlike CaACO4, the RNA expression of these candidates did not follow their respective proteomic trends, suggesting a regulation at the post-translational level (Chapter 7). The identified candidates including CaACO4 are now a resource for further investigation to identify factors that may be involved in capsicum ripening.